Processes and apparatuses for obtaining amniotic mesenchymal stem cells from amniotic fluid and cells derived thereof

ABSTRACT

Methods for purifying, culturing and selecting mesenchymal stem cell (MSC) subpopulations with neonatal quality and adult tissue specificity that are for use in production of advanced therapeutic medicinal products.

FIELD OF THE INVENTION

The present invention relates to methods for purifying, culturing andselecting mesenchymal stem cell (MSC) subpopulations with neonatalquality tissue specificity for use in production of advanced therapeuticmedicinal products.

BACKGROUND OF THE INVENTION

The amniotic fluid is the liquid surrounding and protecting the fetusduring pregnancy. During the last trimester, the amniotic fluid ispartly secreted by the fetal lung and partly by fetal urine. Theamniotic fluid is ingested orally and is absorbed by the gut of thefetus and thus re-enters the fetal circulation. Full term amniotic fluidconsists of water with electrolytes, but also contains proteins,carbohydrates, lipids, phospholipids, and urea. In addition to metabolicwastes, amniotic fluid also contains fetal cells and other materialschafed off the skin such as hair and vernix, a greasy deposit coveringthe skin of a baby at birth. Tissue interfaces in contact with theamniotic fluid contribute to content of the amniotic fluid includingcellular material. The lung is the largest of those surfaces, which alsosecrete lung surfactant into the TAF. The oral and nasal mucosa, theeye, and the urinary tract are other such surfaces with anon-keratinized epithelial interface in topological contact with theamniotic fluid.

Mesenchymal stem cells (MSCs) can be found in nearly all tissues and aremostly located in perivascular niches. As will be understood by one ofskill in the art, mesenchymal stem cells are multipotent stromal cellscapable of differentiating into numerous cell types, and also possessinganti-inflammatory, angiogenic properties for directing tissue repairprocesses, thereby making mesenchymal stem cells valuable fortherapeutic treatments. Term amniotic fluid (TAF) collected during acaesarean section contains a number of valuable cells, including MSCs.However, extracting and growing the MSCs has not previously beenperformed on a large scale due to difficulties associated with sterilelycollecting, handling the TAF and identifying and extracting the MSCs.Moreover, specific subpopulations of MSCs are likely to be particularlywell suited to use for production of therapeutic drugs. Previously, MSCssourced from adult bone marrow, adult adipose tissue or neonatalbirth-associated tissues including placenta, umbilical cord and cordblood were extensively used to obtain MSCs. MSCs from these neonataltissues may have additional capacities in comparison to MSCs derivedfrom adult sources. Indeed, several studies have reported superiorbiological properties such as improved proliferative capacity, life spanand differentiation potential of MSCs from birth-associated tissues overadult derived MSCs. However, neither of these neonatal MSC sources havea corresponding tissue or organ in the adult body. Therefore, a neonatalquality MSCs with tissue specificity would be extremely beneficial.Moreover, acquisition of fetal material may be linked to negativeconsequences for the infant. For example, in cord blood harvesting ithas been shown that as much of the cord blood as possible should bereturned to the infant for improved survival, growth and fine motorskills development. Amniotic fluid, on the other hand, is todayconsidered medical waste that is discarded. Therefore, both the ethicaland practical incentive to harvest such an untapped resource is clear.

SUMMARY OF THE INVENTION

Certain disclosed examples relate to devices, cells, methods, andsystems for obtaining amniotic mesenchymal stem cells from amnioticfluid and cells derived thereof. It will be understood by one of skillin the art that application of the devices, methods, and systemsdescribed herein are not limited to a particular cell or tissue type.Further examples are described below.

In one aspect, the disclosure provides a method for obtaining amnioticmesenchymal stem cells from amniotic fluid, comprising: providing termamniotic fluid (TAF); removing particulate material from the TAF toobtain purified TAF cells; performing adherence selection on thepurified TAF cells to obtain TAF adherence cells; passaging the TAFadherence cells to obtain TAF mesenchymal stem cells (TAF MSCs); andselecting TAF MSCs that express a marker selected from the groupconsisting of TBC1 domain family member 3K (TBC1D3K), allograftinflammatory factor 1 like (AIF1L), cadherin related family member 1(CDHR1), sodium/potassium transporting ATPase interacting 4 (NKAIN4),ATP binding cassette subfamily B member 1 (ABCB1), plasmalemma vesicleassociated protein (PLVAP), mesothelin (MSLN), L1 cell adhesion molecule(L1CAM), hepatitis A virus cellular receptor 1 (HAVCR1), mal, T celldifferentiation protein 2 (gene/pseudogene) (MAL2), SLAM family member 7(SLAMF7), double C2 domain beta (DOC2B), endothelial cell adhesionmolecule (ESAM), gamma-aminobutyric acid type A receptor beta1 subunit(GABRB1), cadherin 16 (CDH16), immunoglobulin superfamily member 3(IGSF3), desmocollin 3 (DSC3), regulator of hemoglobinization anderythroid cell expansion (RHEX), potassium voltage-gated channelinteracting protein 1 (KCNIP1), CD70 molecule (CD70), GDNF familyreceptor alpha 1 (GFRA1), crumbs cell polarity complex component 3(CRB3), claudin 1 (CLDN1), novel transcript (AC118754.1), sodiumvoltage-gated channel alpha subunit 5 (SCN5A), fibroblast growth factorreceptor 4 (FGFR4), potassium two pore domain channel subfamily K member3 (KCNK3), dysferlin (DYSF), ephrin A1 (EFNA1), potassium inwardlyrectifying channel subfamily J member 16 (KCNJ16), membrane associatedring-CH-type finger 1 (MARCHF1), synaptotagmin like 1 (SYTL1),calsyntenin 2 (CLSTN2), integrin subunit beta 4 (ITGB4), vesicleassociated membrane protein 8 (VAMPS), G protein-coupled receptor classC group 5 member C (GPRC5C), CD24 molecule (CD24), cadherin EGF LAGseven-pass G-type receptor 2 (CELSR2), cadherin 8 (CDH8), glutamatereceptor interacting protein 1 (GRIP1), dematin actin binding protein(DMTN), F11 receptor (F11R), cell adhesion molecule 1 (CADM1), cadherin6 (CDH6), coagulation factor II thrombin receptor like 2 (F2RL2),LY6/PLAUR domain containing 1 (LYPD1), solute carrier family 6 member 6(SLC6A6), desmoglein 2 (DSG2), adhesion G protein-coupled receptor G1(ADGRG1), cholecystokinin A receptor (CCKAR), oxytocin receptor (OXTR),integrin subunit alpha 3 (ITGA3), adhesion molecule with Ig like domain2 (AMIGO2), cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1), EPHreceptor B2 (EPHB2).

In another aspect, the disclosure provides isolated cells obtainable bythe method according to the present disclosure, said cells expressing asurface marker selected from the group comprising of TBC1 domain familymember 3K (TBC1D3K), allograft inflammatory factor 1 like (AIF1L),cadherin related family member 1 (CDHR1), sodium/potassium transportingATPase interacting 4 (NKAIN4), ATP binding cassette subfamily B member 1(ABCB1), plasmalemma vesicle associated protein (PLVAP), mesothelin(MSLN), L1 cell adhesion molecule (L1CAM), hepatitis A virus cellularreceptor 1 (HAVCR1), mal, T cell differentiation protein 2(gene/pseudogene) (MAL2), SLAM family member 7 (SLAMF7), double C2domain beta (DOC2B), endothelial cell adhesion molecule (ESAM),gamma-aminobutyric acid type A receptor beta1 subunit (GABRB1), cadherin16 (CDH16), immunoglobulin superfamily member 3 (IGSF3), desmocollin 3(DSC3), regulator of hemoglobinization and erythroid cell expansion(RHEX), potassium voltage-gated channel interacting protein 1 (KCNIP1),CD70 molecule (CD70), GDNF family receptor alpha 1 (GFRA1), crumbs cellpolarity complex component 3 (CRB3), claudin 1 (CLDN1), novel transcript(AC118754.1), sodium voltage-gated channel alpha subunit 5 (SCN5A),fibroblast growth factor receptor 4 (FGFR4), potassium two pore domainchannel subfamily K member 3 (KCNK3), dysferlin (DYSF), ephrin A1(EFNA1), potassium inwardly rectifying channel subfamily J member 16(KCNJ16), membrane associated ring-CH-type finger 1 (MARCHF1),synaptotagmin like 1 (SYTL1), calsyntenin 2 (CLSTN2), integrin subunitbeta 4 (ITGB4), vesicle associated membrane protein 8 (VAMPS), Gprotein-coupled receptor class C group 5 member C (GPRC5C), CD24molecule (CD24), cadherin EGF LAG seven-pass G-type receptor 2 (CELSR2),cadherin 8 (CDH8), glutamate receptor interacting protein 1 (GRIP1),dematin actin binding protein (DMTN), F11 receptor (F11R), cell adhesionmolecule 1 (CADM1), cadherin 6 (CDH6), coagulation factor II thrombinreceptor like 2 (F2RL2), LY6/PLAUR domain containing 1 (LYPD1), solutecarrier family 6 member 6 (SLC6A6), desmoglein 2 (DSG2), adhesion Gprotein-coupled receptor G1 (ADGRG1), cholecystokinin A receptor(CCKAR), oxytocin receptor (OXTR), integrin subunit alpha 3 (ITGA3),adhesion molecule with Ig like domain 2 (AMIGO2), cadherin EGF LAGseven-pass G-type receptor 1 (CELSR1), EPH receptor B2 (EPHB2).

In certain examples, a method for obtaining term amniotic fluidmesenchymal stem cells (TAF MSCs) from term amniotic fluid may comprise:

-   -   providing term amniotic fluid (TAF);    -   removing particulate material from the TAF to obtain purified        TAF cells;    -   performing adherence selection on the purified TAF cells to        obtain TAF adherence cells;    -   passaging the TAF adherence cells to obtain a population of        cells comprising the TAF MSCs; and    -   selecting the TAF MSCs from the population as cells that express        at least one Group A surface marker selected from the group        consisting of TBC1 domain family member 3K, allograft        inflammatory factor 1 like, cadherin related family member 1,        sodium/potassium transporting ATPase interacting 4, ATP binding        cassette subfamily B member 1, plasmalemma vesicle associated        protein, mesothelin, L1 cell adhesion molecule, hepatitis A        virus cellular receptor 1, mal, T cell differentiation protein 2        (gene/pseudogene), SLAM family member 7, double C2 domain beta,        endothelial cell adhesion molecule, gamma-aminobutyric acid type        A receptor beta1 subunit, cadherin 16, immunoglobulin        superfamily member 3, desmocollin 3, regulator of        hemoglobinization and erythroid cell expansion, potassium        voltage-gated channel interacting protein 1, CD70 molecule, GDNF        family receptor alpha 1, crumbs cell polarity complex component        3, claudin 1, novel transcript sodium voltage-gated channel        alpha subunit 5, fibroblast growth factor receptor 4, potassium        two pore domain channel subfamily K member 3, dysferlin, ephrin        A1, potassium inwardly rectifying channel subfamily J member 16,        membrane associated ring-CH-type finger 1, synaptotagmin like 1,        calsyntenin 2, integrin subunit beta 4, vesicle associated        membrane protein 8, G protein-coupled receptor class C group 5        member C, CD24 molecule, cadherin EGF LAG seven-pass G-type        receptor 2, cadherin 8, glutamate receptor interacting protein        1, dematin actin binding protein, F11 receptor, cell adhesion        molecule 1, cadherin 6, coagulation factor II thrombin receptor        like 2, LY6/PLAUR domain containing 1, solute carrier family 6        member 6, desmoglein 2, adhesion G protein-coupled receptor G1,        cholecystokinin A receptor, oxytocin receptor, integrin subunit        alpha 3, adhesion molecule with Ig like domain 2, cadherin EGF        LAG seven-pass G-type receptor 1, and EPH receptor B2, thereby        obtaining the TAF MSCs.

In some examples, selecting TAF MSCs may comprise selecting TAF MSCsthat have a reduced expression of markers selected from the groupconsisting of IL13RA2, CLU, TMEM119, CEMIP, LSP1, GPNMB, FAP, CRLF1,MME, CLMP, BGN, DDR2. Removing particulate matter may comprise filteringand centrifuging the TAF. Performing adherence selection on the purifiedTAF cells may comprise adhering the purified TAF cells to a surfacecoated with a vitronectin-based substrate. The selecting step may beperformed using fluorescence activated cell sorting (FACS). Theselecting step may be performed with antibodies directed to any of themarkers or surface markers. The selecting step may comprise selectingTAF MSCs that express at least two markers from the Group A surfacemarkers. The selecting step may comprise selecting TAF MSCs that expressat least three markers from the Group A surface markers. The selectingstep may comprise selecting TAF MSCs that express at least four markersfrom the Group A surface markers. The selecting step may comprise aplurality of sorting steps, each sorting step comprising directing TAFMSCs into a first output group or a second output group in dependence ona set of markers expressed or not expressed by the respective TAF MSCs.

In some examples, the selecting step may comprise a first sorting stepto direct TAF MSCs that express a Group A surface marker into a firstoutput group, and a second sorting step to direct TAF MSCs from thefirst output group that express a second set of markers into a secondoutput group.

In certain examples, a method for obtaining term amniotic fluid lungmesenchymal stem cells (lung TAF MSCs) from term amniotic fluid, maycomprise:

-   -   providing term amniotic fluid (TAF);    -   removing particulate material from the TAF to obtain purified        TAF cells;    -   performing adherence selection on the purified TAF cells to        obtain TAF adherence cells;    -   passaging the TAF adherence cells to obtain a population of        cells comprising the lung TAF MSCs; and    -   selecting the TAF lung MSCs from the population as cells that        express at least one Group B surface marker selected from the        group consisting of PCDH19, DDR1, MME, IFITM10, BGN, NOTCH3,        SULF1, TNFSF18, BDKRB1, FLT1, PDGFRA, TNFSF4, UNC5B, FAP, CASP1,        CD248, DDR2, PCDH18, LRRC38, and CRLF1, thereby obtaining TAF        lung mesenchymal stem cells.

Selecting lung TAF MSCs may comprise excluding MSCs that express amarker selected from the group consisting of CD24, ITGB4, TNFSF10,GFRA1, CD74, FGFR4, HAVCR1, and OSCAR. The selecting step may compriseselecting TAF MSCs that express at least two surface markers from theGroup B surface markers. The selecting step may comprise selecting TAFMSCs that express at least three surface markers from the Group Bsurface markers. The selecting step may comprise selecting TAF MSCs thatexpress at least four surface markers from the Group B surface markers.The selecting step may comprise selecting TAF MSCs that express asurface marker selected from the group of CD248, DDR1, and LRRC38. Theselecting step may comprise selecting TAF MSCs that express CD248. Theselecting step may comprise selecting TAF MSCs that express CD248 incombination with a marker selected from the group of DDR1 and LRRC38.The selecting step may comprise selecting TAF MSCs that express CD248,DDR1, and LRRC38. In some examples, isolated term amniotic fluid (TAF)mesenchymal stem cells may be obtainable by the methods described above,said cells expressing at least one Group A surface marker.

In some examples, an isolated population of term amniotic fluid (TAF)mesenchymal stem cells, may express at least one Group A surface markerselected from the group comprising of TBC1 domain family member 3K,allograft inflammatory factor 1 like, cadherin related family member 1,sodium/potassium transporting ATPase interacting 4, ATP binding cassettesubfamily B member 1, plasmalemma vesicle associated protein,mesothelin, L1 cell adhesion molecule, hepatitis A virus cellularreceptor 1, mal, T cell differentiation protein 2 (gene/pseudogene),SLAM family member 7, double C2 domain beta, endothelial cell adhesionmolecule, gamma-aminobutyric acid type A receptor beta1 subunit,cadherin 16, immunoglobulin superfamily member 3, desmocollin 3,regulator of hemoglobinization and erythroid cell expansion, potassiumvoltage-gated channel interacting protein 1, CD70 molecule, GDNF familyreceptor alpha 1, crumbs cell polarity complex component 3, claudin 1,novel transcript sodium voltage-gated channel alpha subunit 5,fibroblast growth factor receptor 4, potassium two pore domain channelsubfamily K member 3, dysferlin, ephrin A1, potassium inwardlyrectifying channel subfamily J member 16, membrane associatedring-CH-type finger 1, synaptotagmin like 1, calsyntenin 2, integrinsubunit beta 4, vesicle associated membrane protein 8, G protein-coupledreceptor class C group 5 member C, CD24 molecule, cadherin EGF LAGseven-pass G-type receptor 2, cadherin 8, glutamate receptor interactingprotein 1, dematin actin binding protein, F11 receptor, cell adhesionmolecule 1, cadherin 6, coagulation factor II thrombin receptor like 2,LY6/PLAUR domain containing 1, solute carrier family 6 member 6,desmoglein 2, adhesion G protein-coupled receptor G1, cholecystokinin Areceptor, oxytocin receptor, integrin subunit alpha 3, adhesion moleculewith Ig like domain 2, cadherin EGF LAG seven-pass G-type receptor 1,and EPH receptor B2.

In some examples, a composition may comprise the isolated population ofterm amniotic fluid (TAF) mesenchymal stem cells described above and apharmaceutically acceptable carrier for the TAF MSCs. An isolated termamniotic fluid (TAF) mesenchymal lung stem cells obtainable by a methoddescribed above may express at least one Group B surface marker selectedfrom the group consisting of PCDH19, DDR1, MME, IFITM10, BGN, NOTCH3,SULF1, TNFSF18, BDKRB1, FLT1, PDGFRA, TNFSF4, UNC5B, FAP, CASP1, CD248,DDR2, PCDH18 and CRLF1. In certain examples, an isolated population ofterm amniotic fluid (TAF) lung mesenchymal stem cells may express atleast one Group B surface marker.

In some examples, a method for obtaining term amniotic fluid kidneymesenchymal stem (kidney TAF MSCs) cells from term amniotic fluid, maycomprise:

-   -   providing term amniotic fluid (TAF);    -   removing particulate material from the TAF to obtain purified        TAF cells;    -   performing adherence selection on the purified TAF cells to        obtain TAF adherence cells;    -   passaging the TAF adherence cells to obtain a population of        cells comprising the TAF kidney MSCs; and    -   selecting the TAF kidney MSCs from the population as cells that        express at least one Group C surface marker selected from the        group consisting of HAVCR1, CD24, CLDN6, ABCB1, SHISA9, CRB3,        AC118754.1, ITGB6, CDH1, LSR, EPCAM, AJAP1, ANO9, CLDN7, EFNA1,        MAL2, F11R, L1CAM, GFRA1, IGSF3, TNF, MMP7, FOLR1, TGFA, C3,        TNFSF10, PDGFB and WWC1, thereby obtaining TAF kidney MSCs.

In certain examples, an isolated population of term amniotic fluid (TAF)kidney mesenchymal stem cells (kidney TAF MSCs) may express at least oneGroup C surface marker selected from the group consisting of HAVCR1,CD24, CLDN6, ABCB1, SHISA9, CRB3, AC118754.1, ITGB6, CDH1, LSR, EPCAM,AJAP1, ANO9, CLDN7, EFNA1, MAL2, F11R, L1CAM, GFRA1, IGSF3, TNF, MMP7,FOLR1, TGFA, C3, TNFSF10, PDGFB and WWC1. A composition may comprise theisolated population of term amniotic fluid (TAF) kidney mesenchymal stemcells as described above.

In some examples, a method for obtaining term amniotic fluid skinmesenchymal stem cells (skin TAF MSCs) from term amniotic fluid maycomprise:

-   -   providing term amniotic fluid (TAF);    -   removing particulate material from the TAF to obtain purified        TAF cells;    -   performing adherence selection on the purified TAF cells to        obtain TAF adherence cells;    -   passaging the TAF adherence cells to obtain a population of        cells comprising the TAF skin MSCs; and    -   selecting the skin TAF MSCs from the population as cells that        express at least one Group D surface marker selected from the        group consisting of TNFSF18, PCDH19, NCAM2, TNFSF4, CD248, DDR2,        HTR2B, PCDH18, SULF1, MME, ADGRA2, DCSTAMP, PDGFRA, UNC5B,        SCUBE3, CEMIP, BDKRB1, FLT1, BDKRB2, FAP, CASP1, and SRPX2; and

obtaining TAF skin MSCs.

In certain examples, an isolated population of term amniotic fluid (TAF)skin mesenchymal stem cells (skin MSCs) may express at least one Group Dsurface marker selected from the group consisting of TNFSF18, PCDH19,NCAM2, TNFSF4, CD248, DDR2, HTR2B, PCDH18, SULF1, MME, ADGRA2, DCSTAMP,PDGFRA, UNC5B, SCUBE3, CEMIP, BDKRB1, FLT1, BDKRB2, FAP, CASP1, andSRPX2. A composition may comprise the isolated population of termamniotic fluid (TAF) skin mesenchymal stem cells described above and apharmaceutically acceptable carrier for the TAF skin MSCs.

In some examples, a method for obtaining term amniotic fluid neuralmesenchymal stem cells (neural TAF MSCs) from term amniotic fluid maycomprise:

-   -   providing term amniotic fluid (TAF);    -   removing particulate material from the TAF to obtain purified        TAF cells;    -   performing adherence selection on the purified TAF cells to        obtain TAF adherence cells;    -   passaging the TAF adherence cells to obtain a population of        cells comprising the TAF neural MSCs; and    -   selecting the TAF neural MSCs from the population as cells that        express at least one Group E surface marker selected from the        group consisting of HAVCR1, ACKR3, OSCAR, C3, SIRPB1, SLC6A6,        CCKAR, TNFSF10, CLSTN2, TENM2, SFRP1, PIK3IP1, SCNN1D, CLDN11,        ALDH3B1, and ITGB4; thereby obtaining TAF neural MSCs.

In some examples, an isolated population of term amniotic fluid (TAF)neural mesenchymal stem cells (neural TAF MSCs) may express at least oneGroup E surface marker selected from the group consisting of HAVCR1,ACKR3, OSCAR, C3, SIRPB1, SLC6A6, CCKAR, TNFSF10, CLSTN2, TENM2, SFRP1,PIK3IP1, SCNN1D, CLDN11, ALDH3B1 and ITGB4. A composition may comprisethe isolated population of term amniotic fluid (TAF) neural mesenchymalstem cells described above and a pharmaceutically acceptable carrier forthe TA neural MSCs.

In certain aspects, the disclosure provides methods and apparatuses forisolating term amniotic fluid (TAF) mesenchymal stem cells andcompositions including TAF mesenchymal stem cells comprising one or morefeatures of the foregoing description and/or figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram showing the steps in the purification,culturing and selection of MSC subpopulations.

FIG. 2 is a diagram illustrating a method for collecting amniotic fluid.

FIG. 3 is a schematic illustration, in a perspective view, of anapparatus for filtering amniotic fluid according to an example.

FIG. 4 is a schematic illustration, in a cross-sectional side view, ofan apparatus for filtering amniotic fluid according to an example.

FIG. 5 is a schematic illustration, in a cross-sectional side view, ofan apparatus for filtering amniotic fluid according to an example.

FIG. 6 is a schematic illustration, in a cross-sectional side view, ofan apparatus for filtering amniotic fluid according to an example.

FIG. 7 is a schematic illustration, in a cross-sectional side view, ofan apparatus for filtering amniotic fluid according to an example.

FIG. 8 is a schematic illustration, in a cross-sectional side view, ofan apparatus for filtering amniotic fluid according to an example.

FIG. 9 a schematic illustration, in a cross-sectional side view, of anapparatus for filtering amniotic fluid according to an example.

FIG. 10 is a schematic illustration, in a cross-sectional side view, ofan apparatus for filtering amniotic fluid according to an example.

FIG. 11 is a schematic illustration, in a cross-sectional side view, ofan apparatus for filtering amniotic fluid according to an example.

FIG. 12a is a schematic illustration, in a cross-sectional side view, ofan apparatus for filtering amniotic fluid according to an example.

FIG. 12b is a schematic illustration, along a cross-section A-A in FIG.10a , of an apparatus for filtering amniotic fluid according to anexample.

FIG. 13 is a flow chart of a method of filtering amniotic fluidaccording to an example.

FIG. 14 is a flow chart showing the steps for calculation of an MSCtissue specificity score according to an example.

FIG. 15 is an example graph showing MSC tissue specificity scoresrepresenting the 5% and 15% thresholds.

FIG. 16 is an example graph showing tissue-prioritized and tissue-distaldata, including tissue-prioritized data greater than 15% percentile.

FIGS. 17A-17D show the results of an example study demonstrating theeffects of using TAF Lung MSCs to treat rats with induced lung fibrosis.

DETAILED DESCRIPTION OF THE PREFERRED EXAMPLES

Methods of purifying, culturing and selecting MSC subpopulations withneonatal quality and adult tissue specificity are summarized in FIG. 1and described in detail below. Examples disclosed herein relate toapparatuses and methods for collecting, purifying, isolating, expanding,differentiating, and maturing amniotic fluid-derived cells. The examplesdisclosed herein are not limited to collection of a certain type ofamniotic-derived cell and the technologies disclosed herein are broadlyapplicable to different cells and tissues.

Amniotic Fluid Collection

Amniotic fluid may be collected to produce term amniotic fluid (TAF)according to the methods described in U.S. patent application Ser. No.14/776,499 (corresponding to US2016/0030489), the entire content ofwhich is incorporated by reference. Specifically, FIG. 2 is a blockdiagram of an example of a method 300 of amniotic fluid collection,according to an exemplary example of the invention. It should beappreciated that method 300 may include any number of additional oralternative tasks. The tasks shown in FIG. 3 need not be performed inthe illustrated order, and method 300 may be incorporated into a morecomprehensive procedure or process having additional functionality notdescribed in detail herein.

As shown in FIG. 2, method 300 may include making an incision in theuterine wall 301 of a pregnant mother, for example, during caesareansection. Step 301 may be performed with a standard physician's scalpel.As also shown in FIG. 2, method 300 may include inserting an amnioticfluid collector 302 through the incision in the uterine wall made inStep 301. Method 300 also includes penetrating the amniotic membrane 303using the amniotic fluid collector of Step 302. Step 303 may alsoinclude penetrating the chorionic membrane. In one aspect, the tip isinserted to a 10 cm depth. In some examples, the tip is inserted to adepth of about 3 cm to about 30 cm. In some examples, the tip isinserted to a depth of about 4 cm, about 5 cm, about 6 cm, about 7 cm,about 8 cm, about 9 cm, about 10 cm, about 11 cm, about 12 cm, about 13cm, about 14 cm, about 15 cm, about 16 cm, about 17 cm, about 18 cm,about 19 cm, about 20 cm, about 21 cm, about 22 cm, about 23 cm, about24 cm, about 25 cm, about 26 cm, about 27 cm, about 28 cm, or about 29cm.

Method 300 further includes collecting the amniotic fluid 304 from theamniotic sac using the amniotic fluid collector of Step 302. Step 304may include initiating a siphon to transfer the amniotic fluid to acollection chamber of the amniotic fluid collector, such as by openingan inlet valve of the amniotic fluid collector. Step 304 may alsoinclude positioning a collection chamber of the amniotic fluid collectorbelow an inlet of the amniotic fluid collector. Step 304 may alsoinclude coupling a negative pressure source to an outlet of the amnioticfluid collector to initiate transfer of the amniotic fluid. Step 304 mayinclude relocating an inlet of the amniotic fluid collector to retrievesubstantially all of the available amniotic fluid.

Finally, method 300 includes removing the amniotic fluid collector 905from the amniotic sac. Step 905 may include closing an inlet valve ofthe amniotic fluid collector. In one example, no blood is visible in thecollected material. Step 905 may also include emptying the collectionsystem for further use/processing and sterilizing the exterior of theentire device. In one example, the exterior is sterilized using 70%ethanol so that the sterility may be maintained in any post-processingsteps, such as in a laminar air flow bench setup, e.g., for isolation ofcell material according to the present invention, and for fluid storage.

In one example, the amniotic fluid collection procedure is performed inless than one minute. In one example, the amniotic fluid collectionprocedure is performed in one to two minutes. In one example, theamniotic fluid collection procedure is performed in not more than threeminutes. In one example, the method is simplified compared to standardoperating procedures for cesarean sections, for example, by preventingspillage of the amniotic fluid into the operating wound, improvingvisibility and physical access. In one example, fetal skin is unaffectedby the device tip.

Purification

Term amniotic fluid (TAF) is purified by filtering term amniotic fluidto remove vernix. Although the term ‘term amniotic fluid’ is employedhere and elsewhere in the present disclosure, it is understood thatmethods, processes, and devices of the present disclosure may be appliedto all amniotic fluids and not just term amniotic fluid. Term amnioticfluid may be amniotic fluid collected at term caesarean sectiondeliveries using, for example, a closed catheter-based system. For thepurposes of the present description, ‘tem amniotic fluid’ may beamniotic fluid collected at planned cesarean sections after 37 completedweeks of pregnancy or later, or at planned cesarean section close toterm, for example after 36 completed weeks of pregnancy. Preferably,term amniotic fluid is taken at planned caesarean sections during week37 of pregnancy or later.

FIG. 3 is a schematic illustration of an apparatus 100 for filteringamniotic fluid according to one example. The amniotic fluid containsamniotic cells originating from the fetus or the amniotic sac such asMesenchymal stem cells. The amniotic fluid also contains other materialschafed off the skin such as hair and vernix. Material other than theamniotic cells are here referred to as particulate matter and may alsocomprise meconium, blood clots, etc. Particulate matter may beconsidered as anything larger than 20 μm. For the purposes of filtering,it may be particularly advantageous to treat anything larger than 30 μmor even 50 μm as particulate matter. Optionally, anything larger thanthe targeted amniotic cells may be treated as particulate matter. Theamniotic fluid thus generally contains a mixture of amniotic cells andparticulate matter. The apparatus 100 comprises a filter 101 forfiltering the particulate matter from the amniotic fluid, and a chamber102 enclosing the filter 101. The chamber 102 comprises a fluid inlet103 and a fluid outlet 104. The chamber 102 enclosing the filter 101should be construed as the filter 101 being isolated by the chambertowards the environment surrounding the chamber 102 such that there isno fluid communication between the amniotic fluid in the chamber 102with said environment. Fluid communication through the chamber 102 isthus controlled via the fluid inlet 103 and the fluid outlet 104 in theexample of FIG. 3. The filter 101 is attached to the inside of thechamber 102 between the fluid inlet 103 and the fluid outlet 104. FIG.12 shows an example of a cross-section A-A as indicated in FIG. 12 of acircular chamber 102 and filter 101. It should however be understoodthat the chamber 102 and filter 101 may have varying shapes foroptimization to different applications. The apparatus 100 comprises aninlet connector 105 arranged to form a sealing connection between thefluid inlet 103 and an amniotic fluid sample source 201 (shown in FIG.4). FIG. 4 shows a schematic example of such source 201 of amnioticfluid. Having an inlet connector 105 connected to the fluid inlet 103and configured to provide a sealing connection between the fluid inlet103 directly to a source 201 of amniotic fluid provides for minimizingexposure to contaminants and an efficient aseptic handling of theamniotic fluid. This facilitates obtaining amniotic cells which allowspost-filtration processing at an improved quality standard. Hence, anaseptic pharmaceutical production process is facilitated. Thepreparation of e.g. surfactant molecules may be facilitated. Theapparatus 100 provides for improving the functioning of the amnioticstem cells, such as an improved engraftment phase followingtransplantation. Such improved processes are enabled by having thefilter 101 enclosed in a chamber 102 and an inlet connector 105 arrangedto form a sealing connection between the fluid inlet 103 of the chamber102 and an amniotic fluid sample source 201. The risk of exposing theamniotic stem cells to contaminants, such as bacteria and viruses, isthus reduced. Exposure to oxygen is also minimized, which provides forreducing formation of oxygen free radicals which may negatively impactthe functioning of the stem cells.

FIG. 3 shows an example where the inlet connector 105 comprises a tube105 connected to the fluid inlet 103 at a first sealing connection 114.The inlet connector 105 may form a sealing connection with the fluidinlet 103 with a force-fitting connection, an adhesive, a clamp, orother fixation elements. In another example, such as schematically shownin FIG. 4, the inlet connector 105 is a continuous extension of thefluid inlet 103, without a separate fixation element, e.g. by beingformed as a single piece by molding or other material formingtechniques. FIGS. 3 and 4 show a second connector 115 configured to forma sealing connection with a sample source 201, such as a container orbag 201 containing amniotic fluid. The second connector 115 may comprisereleasable force-fitting connection, a clamp, or a combination thereof,or other releasable fixation elements. The chamber 102, filter 101,fluid inlet 103, fluid outlet 104, and inlet connector 105 may beprovided as a kit in a sterile packaging, e.g. as a disposable kit. Suchkit, i.e. apparatus 100, thus provides for a facilitated and improvedprocess of filtering and obtaining amniotic stem cells. Hence, in use,the amniotic fluid passes the filter 101 when flowing from the fluidinlet 103 to the fluid outlet 104. The particulate matter is thusdeposited on the filter 101 and the amniotic fluid containing theamniotic cells flows through the fluid outlet 104. As seen in theexample in FIG. 12, the filter 101 may be connected around its periphery116 to the inner wall 113 of the chamber 102. This avoids passing ofamniotic fluid from the inlet 103 to the outlet 104 without beingfiltered. The filter 101 may be tensioned or otherwise supported so thata folding or curving of the filter 101 in the chamber 102 is avoided.This maintains a defined mesh or pore size across the area of the filter101 and thus defined filtering characteristics. Maintaining a definedmesh or pore size also reduces the risk of clogging the filter 101.Long-term performance may accordingly be improved.

The apparatus 100 may comprise an outlet 5 connector 106 to form asealing connection between the outlet and an amniotic cell-receivingdevice 202, such as a centrifuge or other amniotic cell-processingequipment downstream of the apparatus 100. FIG. 4 shows a schematicexample of such device 202. This minimizes exposure to contaminants andallows efficient aseptic handling of the amniotic fluid inpost-filtering processing steps. FIG. 3 shows an example where theoutlet connector 106 comprises a tube 106 connected to the fluid outlet104 at a first sealing connection 117. The outlet connector 106 may forma sealing connection with the fluid outlet 104 with a force-fittingconnection, an adhesive, a clamp, or other fixation elements. In anotherexample, such as schematically shown in FIG. 4, the outlet connector 106is a continuous extension of the fluid outlet 104, without a separatefixation element, e.g. by being formed as a single piece by molding orother material forming techniques. FIGS. 3 and 4 show a second connector118 configured to form a sealing connection with an amnioticcell-processing device downstream of the apparatus 100, such as acentrifuge 202. The second connector 118 may comprise a force-fittingconnection, a clamp, a combination thereof, or other releasable fixationelements. The connection between the second connector 118 and e.g. acentrifuge 202 may thus be repeatedly connected and disconnected, andalso re-sealable to maintain a sealing connection in such procedure. Thechamber 102, filter 101, fluid inlet 103, fluid outlet 104, inletconnector 105, and outlet connector 106 may be provided as a kit in asterile packaging, e.g. as a disposable kit. Such kit, i.e. apparatus100, thus provides a facilitated and improved process of filtering andprocessing of amniotic stem cells. The apparatus 100 may comprise a pump122, 123, arranged to pressurize the amniotic fluid to flow from thefluid inlet 103 to the fluid outlet 104. This provides for a moreeffective filtering of the amniotic fluid. Larger volumes may befiltered in less time.

FIG. 6 shows an example where a pump 122 is connected to the fluidoutlet 104 to draw amniotic fluid through the filter 101 in thedirection of the indicated arrows. The pump 122 may be arranged at thefluid inlet 103 to push the amniotic fluid through the filter 101. Thepump 122 may be a compact manually operated pump integrated with thefluid inlet 103, fluid outlet 104, inlet connector 105, or outletconnector 106.

FIG. 7 shows another example, described in more detail below, where apump 123 is arranged to pressurize the amniotic fluid to flow from thefluid inlet 103 to the fluid outlet 104. The chamber 102 may comprise aconduit 119 arranged between the fluid inlet 103 and the fluid outlet104. The pressure in the chamber 102 may be variable in response tofluid and/or gaseous communication through the conduit 119. The flow ofamniotic fluid through the filter 101 may thus be optimized depending onthe application, e.g. the flow rate through the filter 101 may beincreased or decreased by varying the pressure in the chamber 102 viaconduit 119.

FIG. 5 shows an example in which a conduit 119 is in communication withthe chamber 102. An access port 120, such as a connector or valveelement, may be actuated to allow a fluid or gas to be expelled from thechamber 102, and/or injected into the chamber 102, to affect thepressure therein. The conduit 119 is arranged between the fluid outlet103 and the filter 101 in FIG. 5, but the conduit 119 may be arrangedbetween the fluid inlet 103 and the filter 101 in another example. FIG.5 as described below shows a further example of a conduit 119 incommunication with the chamber 102. A pump 123 may be arranged incommunication with the conduit 119, as exemplified in FIG. 7. Thisfacilitates optimization of the flow in the chamber 102 and theassociated filtering process. In the example of FIG. 7 the conduit 119is in variable communication with an upstream cavity 108 of the chamber102 and a downstream cavity 109 of the chamber 102, i.e. the filter 101may be arranged to divide the chamber 102 into an upstream cavity 108and a downstream cavity 109. In FIG. 7 the conduit 119 is connected toboth the upstream cavity 108 and the downstream cavity 109. The pump 123is arranged to pressurize the amniotic fluid to flow from the upstreamcavity 108 to the downstream cavity 109, or to flow from the downstreamcavity 109 to the upstream cavity 108. The latter case may beadvantageous in a situation in which a momentary reversed flow isdesired, e.g. to clear out clogging or occlusion of the filter 101. Insuch case, valves 120, 120′, 121, 121′, as schematically indicated inFIG. 7 are operated to provide the desired flow directions. E.g. for areversed flow, valves 120 and 121′ may be open and valves 120′ and 121may be closed. Valves 121, 121′, may be open and\valves 120, 120′, maybe closed in a normal filtering mode. The upstream cavity 108 may bepressurized by also opening valve 120′ in such filtering mode.

The filter 101 may comprise a first filter element 101 a and a secondfilter element 101 b arranged between the first filter element 101 a andthe fluid outlet 104, as schematically shown in FIG. 8. The secondfilter element 101 b may have a mesh or pore size which is smaller thana mesh or pore size of the first filter element 101 a. This allowseffective filtering of particulate matter of gradually smallerdimensions. The risk of filter occlusion is thus reduced. This allowsfor a more reliable and robust filtering process of the amniotic fluid.An improved filtering of amniotic fluid containing a greater range inthe size of particulate matter is also provided. Further, a largerfraction of the stem cells in the amniotic fluid may be obtained sincethe stem cells are not lost in clogged pores. Although FIG. 8 two filterelements 101 a, 101 b, it should be understood that any plurality offilter elements may be arranged in sequence in the chamber 102, withgradually decreasing mesh or pore size, in the direction of fluid flowfrom the fluid inlet 103 to the fluid outlet 104, for an effectivefiltering of particulate matter of gradually decreasing dimensions. Thefirst and second filter elements 101 a, 101 b, may be separated by adistance (d) along a direction amniotic fluid flow from the fluid inlet103 to the fluid outlet 104, as schematically indicted in the example ofFIG. 8. The motion of the amniotic fluid between the first and secondfilter elements 101 a, 101 b, which in some case may involve turbidflow, may provide for further reducing the risk of unwanted build-up ofparticles on the first and second filter elements 101 a, 101 b.

The filter 101 may comprise a mesh having a mesh size in the range of20-2000 μm. In another example, the filter 101 comprises a mesh having amesh size in the range of 100-500 μm. This allows particularly effectivefiltration of particulate matter from the amniotic fluid. Turning againto FIG. 8, the first filter element 101 a may comprise a mesh having amesh size in the range of 500-1000 μm, and the second filter element 101b may comprise a mesh having a mesh size in the range of 30-150 μm. Thefirst filter element 101 a may thus remove larger debris, followed byremoval of smaller particles with the second filter element 101 b. Thisallows a particularly effective filtering of particulate matter ofvarying size and reliable filtering of increased volumes over longertime periods since the risk of clogging is further minimized. Aspreviously mentioned, any plurality of filter elements may be arrangedin succession in the chamber 102.

FIG. 9 shows three filter elements 101 a, 101 b, 101 c, arranged in thechamber 102. In some examples the filter element having the smallestmesh or pore size, arranged furthest downstream in the chamber 102 may,such as filter element 101 b in FIG. 6 and filter element 101 c in FIG.9, may have a mesh or pore size dimensioned so that only single amnioticcells or amniotic cell clumps smaller than 10 cells pass through thefilter 101. The smallest mesh or pore size in such an example may beapproximately 30 μm. The filter 101 may comprise a mesh such as a nylonmesh. The filter 101 may comprise a porous material having a variablepore size through the filter 101 in the direction of flow of theamniotic fluid from the fluid inlet 103 to the fluid outlet 104. I.e.larger debris is removed at the surface of the filter 101 closest to theinlet 103 whereas particles of smaller size are removed deeper into thefilter, as the amniotic fluid flows through the filter 101 in adirection towards the outlet 104 and the size of the pores get smaller.As previously mentioned, the chamber 102 may comprise an upstream cavity108 and a downstream cavity 109. The upstream and downstream cavities108, 109, may be formed as an integrated piece to form the chamber 102,e.g. in a molding process or by other material forming techniques. Theupstream and downstream cavities 108, 109, may be formed as separateunits which are then connected to each other to form a sealingconnection, e.g. by an adhesive or by welding. The filter 101 may beattached simultaneously or subsequently with such welding process or bythe aforementioned adhesive.

The upstream and downstream cavities 108, 109, may be releasablyconnectable to each other at a connecting element 110, to form a sealingconnection, as schematically shown in FIG. 9. This allows opening of thechamber 102, e.g. for replacing the filter 101. The filter 101 may thusbe releasably connectable to the chamber 102, e.g. filter elements 101a, 101 b, 101 c, may be releasably connectable to the chamber 102 inFIG. 7. This allows facilitated customization to different applicationssince filter elements 101 a, 101 b, 101 c, of different pore or meshsize, or different number of such filter elements may be mounted in thechamber 102.

The connecting element 110 is configured to form a sealing connectionupstream and downstream cavities 108, 109, and may comprise an annulargasket extending around the periphery of the upstream and downstreamcavities 108, 109. The filter 101 may comprise a cartridge of differentnumbers of filter elements 101 a, 101 b, 101 c, with different poresizes that could be tailored to the particular amniotic fluid sample.For example, evaluation of the amniotic fluid turbidity and degree ofmilkiness (level of vernix both in particle size and opaqueness) couldbe an indicator of the appropriate filter cartridge to use. Anaccompanying chart for which to compare the amniotic fluid sample withcould indicate which filter cartridge to use. The upstream cavity 108and/or the downstream cavity 109 may be funnel shaped. FIGS. 3-9 showexamples where both the upstream and downstream cavities 108, 109, arefunnel shaped. FIG. 11 shows an example where only the downstream cavity109 is funnel shaped. Having a funnel shape may be advantageous fordirecting the flow of amniotic fluid along a desired vector of symmetrythrough the filter 101 and apparatus 100. The upstream cavity 108 and/orthe downstream cavity 109 may comprise a chamber wall 111 a, 111 b beingarranged essentially in parallel with the filter 101, i.e. perpendicularto the direction of flow of the amniotic fluid from the fluid inlet 103to the fluid outlet 104. FIG. 10 shows an example where chamber walls111 a, 111 b, of the upstream and downstream cavities 108, 109 arearranged essentially in parallel with the filter 101. This minimizes thespace inside the chamber 102, while maintaining adequate filter area, tominimize the risk of introducing e.g. air that may disturb surfactantmolecules, reduce the risk of infection, and reduce detrimentalformation of reactive oxygen species in the amniotic cells. The chamber102, and/or the inlet connector 105, and/or the outlet connector 106 maybe formed from a phthalate free PVC material. This provides for anapparatus which is suitable to be in contact with pharmaceuticalstarting materials such as amniotic cells.

The apparatus 100 may comprise protrusions 112 arranged to extend froman inner wall 113 of the chamber 102. FIGS. 11 and 12 show examples ofsuch protrusions 112, in a cross-sectional side view and throughcross-section A-A respectively. The protrusions 112 provides support forthe filter 101 in case the filter 101 would start bend and fold towardsthe inner wall 113. Thus, a flow through the mesh or pores of the filter101 is still possible in such case since the filter 101 may be supportedby the protrusions 112 at a distance from the inner wall 113, i.e. theprotrusions 112 allows for further limiting the risk of flow restrictionand provides for an efficient, robust and reliable filtering.

FIG. 13 is a flow chart of a method 300 of filtering amniotic fluidcontaining particulate matter and amniotic cells. The method 300comprises forming 301 a sealing connection between a fluid inlet 103 ofa chamber 102 and an amniotic fluid sample source 201. The method 300comprises passing 302 the amniotic fluid through a filter 101 enclosedin the chamber 102 by providing a flow of the amniotic fluid from thefluid inlet 103 to a fluid outlet 104 of the chamber 102. Particulatematter is thereby deposited on the filter 101 and the amniotic fluidcontaining amniotic cells flows through the outlet 104. The method 300thus provides for the advantageous benefits as described in relation toapparatus 100 and FIGS. 3-12 above. The method 300 provides foreffective and sterile filtration of the amniotic fluid to obtainamniotic cell samples of high quality.

In one embodiment, removing particulate material from the TAF to obtainpurified TAF cells may be done by applying any known method in the artsuch as filtration, centrifugation, etc. The TAF may be filtered througha filter having a pore size at or above 20 μm. The filter may be madefrom any synthetic material including but not limited to celluloseacetate, cellulose nitrate (collodion), polyamide (nylon),polycarbonate, polypropylene and polytetrafluoroethylene (Teflon). Inone embodiment removing particulate material is done by applyingapparatus 100.

Adherence Selection

Various terms known to one skilled in the art have been and will be usedthroughout the specification, for example, the terms “express,expression, and/or expressing” in the context of a cell surface markerare meant to indicate the presence of a particular marker on the surfaceof a cell, said surface marker having been produced by the cell. Surfacemarker expression may be used to select between different cellpopulations, for example, positively selecting for surface markerexpression indicates the selection of a cell population that morestrongly expresses a particular surface marker as compared to anothercell population. Conversely, negatively selecting for cell surfacemarker expression indicates the selection of a cell population that moreweakly expresses a particular surface marker as compared to another cellpopulation.

As explained above and elsewhere in the specifications, TAF containsvarious progenitor cell types. In certain examples, particularprogenitor cell types may be isolated and propagated via adherenceselection. For example, a vitronectin substrate, Synthemax (Merck,CORNING®, Synthemax®, II-SC SUBSTRATE, CLS3535-1EA) may be used as acoating to create a more in vivo-like environment for stem cell culture,thereby limiting maturation of the TAF-derived progenitor cells andmaintaining plasticity. Synthemax is an animal-component free,synthetic, flexible vitronectin-based peptide substrate for serum orserum-free expansion of human progenitor/stem cells and other adult stemcell types. One of skill in the art will understand that thevitronectin-based peptide substrate may include a portion of avitronectin protein, such as a particular peptide sequence ofvitronectin. Alternatively, intact vitronectin protein may be used.Synthemax vitronectin substrate offers a synthetic, xeno-freealternative to biological coatings and/or feeder cell layers commonlyused in cell culture and known in the art. Briefly, standardtissue-culture treated flasks may be coated with about 0.2 mLSynthemax/cm² at 10 μg/mL giving a surface density of 2 μg/cm², andincubated at 37° C. for about 1 h, 0.5 h 2 h, 4 h, 8 h, or more than 8 hor at room temperature for about 2 h, 1 h, 4 h, 8 h or more than 8 hwith surplus solution optionally being removed and replaced. In certainexamples, Synthemax may be coated at a surface density of about: 1 to 5μg/cm², such as 2 μg/cm², 0.1 to 10 μg/cm², 0.5 to 4 μg/cm², 1 to 3μg/cm², or about 1.5 to 2.5 μg/cm².

In other embodiments, adherence selection can be performed using asurface coated with, for example, Collagen, Fibronectin. Alternatively,adherence selection can be performed using an uncoated surfacecomprising a tissue-culture treated plastic.

Cells purified from TAF fluid may be gently re-suspended in prewarmedxeno-free cell culture media, with the cell suspension is then added tothe Synthemax-coated flasks. Media may be changed at various times afteraddition to the flasks, for example, after about: 2 h to 168 h, 12 h to96 h, 24 h to 72 h, 36 h to 60 h, 42 h to 56 h, or 48 h, and thensubsequently changed about: every day, every other day, every third day,every fifth day, once a week, once every two weeks or about less thanonce every two weeks. Through repeated removal of spent medium, thenon-attached cells may be removed, thereby selecting the MSCs by theiraffinity for attachment to the Synthemax-treated surface. The cells maybe cultured for a period of time, such as about, for example, 4 d, 7 d,10 d, 11 d, 12 d, 13 d, 14 d, 18 d, 21 d, 28 d or longer than 21 d.Optionally, in some examples, the cells may be cultured under hypoxicconditions, hypoxia priming may alter cell metabolism during expansion,increase resistance to oxidative stress, and thereby improve theengraftment, survival in ischemic microenvironments, and angiogenicpotential of transplanted MSCs. After culturing, the PO colonies (Colonyforming Units—CFUs) that have formed may be dissociated and pooled.After pooling, the remaining cells may be predominantly non-tissuespecific MSCs. In certain examples, the pooled PO cells may be gentlyre-suspended in pre-warmed xeno-free cell culture media and re-plated ontissue-culture treated flasks without Synthemax for passaging. Thepooled cells may be seeded at a seeding density of from between about:100 to 10000 cells/cm², 500 to 8000 cells/cm², 1000 to 5000 cells/cm²,or about 2000 to 4000 cells/cm². The media may be changed about every 1d, 2 d, 4 d, or more than four days. After a period of time, such asabout 2 d, 4 d, 7 d, or more than 7 d, the cells may be dissociated andharvested. Further selective MSC isolation may be achieved as describedbelow.

Identification of Markers

When comparing the genetic expression profiles of TAF-MSCs andadult-type MSCs derived from adipose tissue or bone marrow by RNAseq,TAF-MSCs tend to express more of some genes present in adult-type MSCsand less of others. Identification of both positive and negative TAF-MSCspecific neonatal cell-surface markers can allow for sorting of the MSCswith neonatal quality from those that have differentiated further andare of less importance as progenitor cells using e.g. ligands such asantibodies and aptamers or other selection techniques.

The cell surface markers distinguishing tissue relevant cells from otherMSCs may be elucidated via a bioinformatics process utilizing atissue-specificity score algorithm. An example of an MSCtissue-specificity score algorithm is shown in FIG. 14.Tissue-specificity may be measured as a combination of two components: a‘tissue transcriptional similarity’ also known as a similarity score anda “tissue-specific gene expression program” also known as a gene setscore. In certain examples, the similarity score may be an AverageSpearman correlation to each MSC tissue reference sample (for example afetal lung MSC sample). In examples, the gene set score may be theaverage expression of genes in a tissue-specific gene set. As shown inFIG. 14, in certain examples, after normalizing the similarity and geneset scores using a Z-transform to convert the input values, which is asequence of real or complex numbers, into a complex frequency-domainrepresentation, then combining them assigning equal weight to each scoreand transforming combined values using a Z-transform, the resultingoutput is an MSC tissue specificity score. The MSC tissue-specificityscore measures the relative tissue-specificity among the input samplesby measuring how many standard deviations a sample is more or lessspecific to a given tissue compared to the average input sample. Forexample, an MSC tissue-specificity score may indicate how much more aclone sample appears to have a tissue specific phenotype, such as a lungphenotype, as compared to an average clone. Such an approach allows foridentification of the top X % percentile scores using a normaldistribution function, effectively the top X % of clones that are mosttissue-specific to the relevant tissue.

In one example, for a given tissue, tissue-prioritized clones can bedefined as any clone belonging to the top X % percentile score, where Xis any percentage within a range having a lower end from about 0.1 to25, such as about 1, 5, 10, 15 and 20, and an upper end from about 30 to75, such as about: 35, 40, 45, 50, 55, 60, 65 or 70. An example ofTAF-MSC tissue-specificity prioritization results is shown in FIG. 15,in which thresholds at 15% and 5% are visible. Having prioritizedtissue-specific clones, candidate surface marker genes may then beidentified. For each tissue, two groups may be defined:tissue-prioritized and tissue-distal. A suitable analysis program may beused to make this determination, for example DEseq2 fromBioconductor.org. The tissue-prioritized group may include clones with ascore in the top 15% percentile. The tissue-distal group may includeclones in the bottom Y % percentile in which Y is any percentage withinthe range having a lower end from about 25 to 70, such as about: 30, 35,40, 45, 50, 55, 60 or 65 and an upper end from 75 to 99.9, such asabout: 80, 85, 90, 95 or 99. FIG. 16 shows an example of such analysison kidney tissue. Next, differentially expressed genes between thetissue-prioritized and tissue-distal groups may be identified. Finally,the differential expression results may be annotated with surface markergene information.

In certain examples, to identify tissue-specific cell surface markers,surface marker genes with a more than a Z-fold increase, where Z is atleast about: 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold,5-fold, 8-fold, 10-fold, 12-fold, 15-fold or even more-fold increase inexpression (log 2FoldChange) in prioritized clones compared to anaverage clone and a Transcripts Per Kilobase Million (TPM) of more thanabout 500, such as more than about: 1000, 1500, 2000, 2500, 3000, 5000or even higher may be selected to give the top tissue-specific markercandidates, such as approximately the top: 5, 10, 20, 30, 40, 50, 60,70, 100 or more, for example such as those shown below in Tables 3-6 andfurther described in more detail below. Suitable log 2FoldChange and TPMvalues may vary even further depending on tissue type specificitiesdepending on the abundance/absence of good markers.

Applying the tissue specificity algorithms described above to identifysurface markers, after adhesion selection and passaging, the TAF-MSCscells may express various identified surface markers as shown below inTable 1, indicative of non-tissue specific TAF MSCs. One of skill in theart will understand that such surface markers may be present at varioussurface densities and may be upregulated or downregulated in comparisonto other cell types. Therefore, such surface markers may be used toidentify and isolate particular cell types. In some instances, thesurface markers listed in Table 1 below may be at least 8-fold morehighly expressed for TAF MSCs on average compared to other MSC celltypes, particularly as compared to adult MSCs derived from bone marrowor adipose tissue. The thresholds used to generate Table 1 are asfollows: X was selected as 15%, Y was selected as 50%, Z was selected as8-fold and a TPM of more 3000 was selected. One of skill in the art willunderstand that the numbering used in Table 1 and all tables herein ismerely used to indicate a total number of identified markers and not toindicate that one particular marker is more strongly expressed and/orpreferred compared to another marker.

TABLE 1 1. TBC1D3K TBC1 domain family member 3K 2. AIF1L allograftinflammatory factor 1 like 3. CDHR1 cadherin related family member 1 4.NKAIN4 sodium/potassium transporting ATPase interacting 4 5. ABCB1 ATPbinding cassette subfamily B member 1 6. PLVAP plasmalemma vesicleassociated protein 7. MSLN mesothelin 8. L1CAM L1 cell adhesion molecule9. HAVCR1 hepatitis A virus cellular receptor 1 10. MAL2 mal, T celldifferentiation protein 2 (gene/ pseudogene) 11. SLAMF7 SLAM familymember 7 12. DOC2B double C2 domain beta 13. ESAM endothelial celladhesion molecule 14. GABRB1 gamma-aminobutyric acid type A receptorbetal subunit 15. CDH16 cadherin 16 16. IGSF3 immunoglobulin superfamilymember 3 17. DSC3 desmocollin 3 18. RHEX regulator of hemoglobinizationand erythroid cell expansion 19. KCNIP1 potassium voltage-gated channelinteracting protein 1 20. CD70 CD70 molecule 21. GFRA1 GDNF familyreceptor alpha 1 22. CRB3 crumbs cell polarity complex component 3 23.CLDN1 claudin 1 24. AC118754.1 novel transcript 25. SCN5A sodiumvoltage-gated channel alpha subunit 5 26. FGFR4 fibroblast growth factorreceptor 4 27. KCNK3 potassium two pore domain channel subfamily Kmember 3 28. DYSF dysferlin 29. EFNA1 ephrin A1 30. KCNJ16 potassiuminwardly rectifying channel subfamily J member 16 31. MARCHF1 membraneassociated ring-CH-type finger 1 32. SYTL1 synaptotagmin like 1 33.CLSTN2 calsyntenin 2 34. ITGB4 integrin subunit beta 4 35. VAMP8 vesicleassociated membrane protein 8 36. GPRC5C G protein-coupled receptorclass C group 5 member C 37. CD24 CD24 molecule 38. CELSR2 cadherin EGFLAG seven-pass G-type receptor 2 39. CDH8 cadherin 8 40. GRIP1 glutamatereceptor interacting protein 1 41. DMTN dematin actin binding protein42. F11R F11 receptor 43. CADM1 cell adhesion molecule 1 44. CDH6cadherin 6 45. F2RL2 coagulation factor II thrombin receptor like 2 46.LYPD1 LY6/PLAUR domain containing 1 47. SLC6A6 solute carrier family 6member 6 48. DSG2 desmoglein 2 49. ADGRG1 adhesion G protein-coupledreceptor G1 50. CCKAR cholecystokinin A receptor 51. OXTR oxytocinreceptor 52. ITGA3 integrin subunit alpha 3 53. AMIGO2 adhesion moleculewith Ig like domain 2 54. CELSR1 cadherin EGF LAG seven-pass G-typereceptor 1 55. EPHB2 EPH receptor B2

As will be understood by one of skill in the art, suitable combinationsof the markers listed in Table 1 may be used to separate TAF-MSCs fromadult MSCs by selecting for specific markers from Table 1 orcombinations of two, three, four, five, six or more markers fromTable 1. In certain examples, TAF MSCs can be more specificallyidentified by identifying a combination of stronger expression, such as8-fold or more stronger expression of any combination of the foregoingmarkers, e.g., TBC1D3K and/or AIF1L and/or CDHR1 and/or NKAIN4 and/orABCB1 and/or PLVAP as compared to adult MSCs. When using combinations ofmarkers, identification may be achieved with a lower threshold ofstronger expression, such as 2-fold or more, 4-fold or more, or 6-foldor more expression of each of the markers.

In contrast to the above surface markers that may be more stronglyexpressed on the surface of TAF-MSCs (positive markers) compared toadult MSCs, in certain examples, the below surface markers in Table 2may be more weakly expressed on TAF-MSCs as compared to other cell types(negative markers), such as ⅛-fold or less expression (optionally withTPM threshold >500) of any combination of the foregoing markers versusadult MSCs: IL13RA2, CLU, TMEM119, CEMIP, and LSP1. When usingcombinations of negative markers, identification may be achieved with alower threshold of weaker expression, such as ½-fold or less, ¼-fold orless, or ⅙-fold or less expression of each of the markers.

Combinations of two or more these negative markers can also be used tomore specifically isolate TAF MSCs. In addition, those skilled in theart will also recognize that combinations including both negative andpositive markers, such as at any of the thresholds described above, canalso be effective to more specifically isolate TAF MSCs.

TABLE 2 1. IL13RA2 Interleukin-13 receptor subunit alpha-2 2. CLUClusterin 3. TMEM119 Transmembrane Protein 119 4. CEMIP Cell MigrationInducing Hyaluronidase 1 5. LSP1 Lymphocyte Specific Protein 1 6. GPNMBGlycoprotein Nmb 7. FAP Fibroblast Activation Protein Alpha 8. CRLF1Cytokine Receptor Like Factor 1 9. MME Membrane Metalloendopeptidase 10.CLMP CXADR Like Membrane Protein 11. BGN Biglycan 12. DDR2 DiscoidinDomain Receptor Tyrosine Kinase 2

Marker-Based Selection

Amniotic fluid contains heterogenous cells in a homogenous fluid. Hence,a marker-based selection may be needed. One example of marker-basedselection is via the use of Fluorescence activated cell sorting (FACS).Fluorescence activated cell sorting (FACS) may be used to purify thecell population of TAF-MSCs, FACS allows for a very high purity of thedesired cell population, even when the target cell type expresses verylow levels of identifying markers and/or separation is needed based ondifferences in marker density. FACS allows the purification ofindividual cells based on size, granularity and fluorescence. As will beunderstood by one of skill in the art, FACS may be used to select forcertain cell populations that express one cell surface marker more thananother cell population and vice-versa. In some examples of methods ofpurification, bulk methods of purification such as panning, complementdepletion and magnetic bead separation, may be used in combination withFACS or as an alternative to FACS. In brief, to purify cells of interestvia FACS, they are first stained with fluorescently-tagged monoclonalantibodies (mAbs), which recognize specific surface markers on thedesired cell population. Negative selection of unstained cells may alsoallow for separation. For GMP production of cells according to someexamples, FACS may be run using a closed system sorting technology suchas MACSQuant® Tyto®. Samples may be kept contamination-free within thedisposable, fully closed MACSQuant Tyto Cartridge. Further, filtered airmay drive cells through a microchannel into the microchip at very lowpressure (<3 PSI). However, before entering the microchannel, potentialcell aggregates may be held back by a filter system guaranteeing asmooth sorting process. The fluorescence detection system may detectcells of interest based on predetermined fluorescent parameters of thecells. Based on their fluorescent and scatter light signatures, targetcells may be redirected by a sort valve located within the microchannel.For certain examples of methods of purification, the success of stainingand thereby sorting may depend largely on the selection of theidentifying markers and the choice of mAb. Sorting parameters may beadjusted depending on the requirement of purity and yield. Unlike onconventional droplet sorters, cells sorted by the MACSQuant Tyto may notexperience high pressure or charge, and may not get decompressed.Therefore, such a gentle sorting approach may result in high viabilityand functionality of cells. Alternatively, other marker-based selectiontechniques may be known to the skilled person and employed here. Theseinclude, but are not limited to, Magnetic-activated cell sorting,Microfluidic based sorting, Buoyancy activated cell sorting, masscytometry etc.

Tissue Specific Cells and Usage Lung TAF Cell Markers

As explained above, analysis of RNAseq data from TAF-MSC clones, adultand neonatal MSC reference material as well as fetal fibroblasts andpublicly available expression datasets may be used to identify andcharacterize TAF-MSC cells. For example, sub-populations of TAF-MSCs maybe established by clustering their expression data (RNAseq) withneonatal reference samples. Such sub-populations include, but are notlimited to, lung MSC, urinary tract MSC (described also as kidney MSCsin the present disclosure), and skin MSC. Gene lists of highly and lowlyexpressed genes for each cluster of expression data may allow foridentification of surface maker genes for each cluster. Using such datacomparison, sub-populations of TAF cells were compared to adult MSCcells based on their gene expressions (RNAseq) resulting in a list ofneonatal-specific surface marker genes for each cluster. A number ofsurface markers of interest associated with lung TAF cells wereidentified. For example, a non-exclusive list of preferred surfacemarkers used to identify and separate lung TAF cells are provided below.Moreover, as the number of different MSC-subtypes in TAF is limited, theselection of the tissue specific MSC may be done by firstlycharacterization, thereafter a stepwise negative selection/sorting ofthe material by taking into account the combined (multivariate) surfacemarker profile of the different tissue specific MSC's. One of skill inthe art will understand that any such combination of these surfacemarkers may be used for identifying and isolation of lung TAF cells fromthe general population of TAF-derived cells and/or TAF-MSC cells. Insome examples, the below non-exclusive list of surface markers may bemore highly expressed on the surface of Lung-TAF cells as compared toother cell types, such as other TAF-derived cells and/or TAF-MSC cells.

As explained above, bioinformatics techniques may be used to identifytissue-specific surface markers, therefore, the surface markersidentified in Table 3 may have at least a 10-fold increase in expressionon prioritized clones compared to the average TAF-MSC clone (optionallywith TPM threshold >2000).

TABLE 3 1. PCDH19—protocadherin 19; 2. DDR1—discoidin domain receptortyrosine kinase 1; 3. MME—membrane metalloendopeptidase; 4.IFITM10—interferon induced transmembrane protein 10; 5. BGN—biglycan; 6.NOTCH3—notch receptor 3; 7. SULF1—sulfatase 1; 8. TNFSF18—TNFsuperfamily member 18; 9. BDKRB1—bradykinin receptor B1; 10. FLT1—fm srelated tyrosine kinase 1; 11. PDGFRA—platelet derived growth factorreceptor alpha; 12. TNFSF4—TNF superfamily member 4; 13. UNC5B—unc-5netrin receptor B; 14. FAP—fibroblast activation protein alpha; 15.CASP1—caspase 1; 16. CD248—Endosialin; 17. DDR2—discoidin domainreceptor tyrosine kinase 2; 18. PCDH18—protocadherin 18; and/or 19.CRLF1—cytokine receptor like factor 1;

In contrast to the above surface markers that may be more stronglyexpressed on the surface of lung TAF MSCs, in certain examples, thebelow surface markers may be more weakly expressed on lung TAF MSCs ascompared to other cell types, such as other TAF-derived cells and/orTAF-MSCs: CD24, ITGB4, TNFSF10, GFRA1, CD74, FGFR4, HAVCR1, and OSCAR.As will be understood by one of skill in the art, one, two, three, four,or more of the aforementioned more weakly expressed surface markers maybe used to separate lung TAF cells from other cell types such as otherTAF-derived cells and/or TAF-MSCs.

In certain examples, the cell surface marker CD248 (Endosialin) may beused to sort lung TAF MSCs from a population of TAF MSCs. Furthersurface markers that may be used to sort lung TAF MSCs include DDR-1(discoidin domain receptor tyrosine kinase 1) as well as LRRC38 (LeucineRich Repeat Containing Protein 38), all three of which have beenidentified via antibodies as useful markers for separation. In someexamples, Endosialin, DDR-1, and/or LRRC38 alone or in combination withother markers may be used to sort. Endosialin may be combined with DDR-1or LRRC38 to sort, or DDR-1 and LRRC38 may be combined withoutEndosialin.

As will be understood by one of skill in the art, suitable combinationsof the markers listed in Table 3 and CD248, DDR-1, and LRR38 may be usedto separate lung TAF MSCs from TAF MSCs by selecting for specificmarkers from Table 3 or combinations of two, three, four, five, six ormore markers from Table 3 and/or CD248 and/or DDR-1 and/or LRR38. Incertain examples, lung TAF MSCs can be more specifically identified byidentifying a combination of stronger expression, such as 10-fold ormore stronger expression (optionally with TPM threshold >2000) of anycombination of the foregoing markers, e.g., PCDH19 and/or DDR1 and/orMME and/or IFITM10 and/or BGN and/or NOTCH3 and/or CD248 and/or DDR-1and/or LRR38 as compared to TAF MSCs. When using combinations ofmarkers, identification may be achieved with a lower threshold ofstronger expression, such as 4-fold or more, 6-fold or more, or 8-foldor more expression of each of the markers.

In contrast to the above surface markers that may be more stronglyexpressed on the surface of lung TAF MSCs (positive markers) compared toTAF MSCs, in certain examples, the below surface markers may be moreweakly expressed on lung TAF-MSCs as compared to other cell types(negative markers), such as ⅛-fold or less expression (optionally withTPM>500) of any combination of the foregoing markers versus TAF MSCs:CD24, ITGB4, TNFSF10, GFRA1, CD74, FGFR4, HAVCR1, and OSCAR. When usingcombinations of negative markers, identification may be achieved with alower threshold of weaker expression, such as ½-fold or less, ¼-fold orless, or ⅙-fold or less expression of each of the markers.

Combinations of two or more these negative markers can also be used tomore specifically isolate lung TAF MSCs. In addition, those skilled inthe art will also recognize that combinations including both negativeand positive markers, such as at any of the thresholds described above,can also be effective to more specifically isolate lung TAF MSCs.

FIGS. 17A-17D show an example of the results from a proof-of-principlestudy on the potential use of Lung TAF MSCs for treatment, performedusing neonatally sorted TAF MSCs expressing MSC lung cell surfacemarkers including CD248, DDR1, and LRRC38 (called “LBX-THX-001 cells”).The purpose of the study was to investigate the effects of LBX-THX-001cells in a bleomycin induced lung fibrosis model in male rats. Two cellconcentrations (2 M cell/kg and 5 M cells/kg) and two types of vehiclesfor the cells were tested (PBS and CryoStor CS-10).

The development of fibrosis in rat lung after exposure to bleomycin iswell documented in the literature and a frequently used model forstudying the pathology of lung fibrosis and also the effect of differenttreatments. The number of LBX-THX-001 cells injected were chosen to berelevant for a possible human therapy. The number of cells weretherefore chosen to reflect cell numbers used in previous studies onrats (8-20 M cells/kg) and humans (0.5-2 M cells/kg).

An intra-tracheal instillation of bleomycin (1000 U/rat) to 34 maleSD-rats was used to induce lung fibrosis in the rats. During the firstweek, the rats were monitored and weighed daily and thereaftertwice/week until termination of the study. At day 4 post bleomycinchallenge, the LBX-THX-001 cells were administered by an intra-venous(i.v.) injection. The injection volume was 194-535 μL (maximal toleratedinjection volume 1 mL/kg). The response to the intra-trachealinstillation of bleomycin was as expected based on previous experiencefor the model with weight loss during the first days after instillationand thereafter recovery. There were no significant differences in weightloss between the bleomycin group and the treatment groups.

As shown in FIGS. 17A-D, bleomycin instillation induced fibrotic changein the lung. The histopathological evaluation concluded pathologicalchanges in the bleomycin group both with regard to percent of parenchymaaffected and after scoring using the modified Ashcroft scale. As shownin FIGS. 17A-D, the group treated with LBX-THX-001 cells (2 millioncells/kg) 4 days post Bleomycin showed significantly less fibrosis intheir lungs compared to the bleomycin group. This was seen both in thehistopathological evaluation using the read-out “percent parenchymaaffected” (FIGS. 17A-B) and the fibrosis scoring Ashcroft modified scale(FIGS. 17A-D). No human MSCs could be detected in rat lungs attermination (after 28 days).

Kidney TAF Cell Markers

Similar to the lung TAF MSC cell markers identified above, a number ofsurface markers of interest associated with kidney TAF cells wereidentified. For example, a non-exclusive list of surface markers used toidentify and separate kidney TAF MSCs are provided below in Table 4.Similar to the lung TAF MSC markers, the surface markers identified inTable 4 may have at least a 12-fold increase in expression onprioritized kidney TAF clones compared to the average TAF-MSC clone(optionally with TPM threshold >2000). Moreover, as the number ofdifferent MSC-subtypes in TAF is limited, the selection of the tissuespecific MSCs may be done first by characterization, and thereafter by astepwise negative selection/sorting of the material by taking intoaccount the combined (multivariate) surface marker profile of thedifferent tissue specific MSC's. One of skill in the art will understandthat any such combination of these surface markers may be used foridentifying and isolation of kidney TAF cells from the generalpopulation of TAF-derived cells and/or TAF-MSC cells. In some examples,the below non-exclusive list of surface markers may be more highlyexpressed on the surface of kidney-TAF cells as compared to other celltypes, such as other TAF-derived cells and/or TAF-MSC cells:

TABLE 4 1. HAVCR1—hepatitis A virus cellular receptor 1; 2. CD24—CD24molecule; 3. CLDN6—claudin 6; 4. ABCB1—ATP binding cassette subfamily Bmember 1; 5. SHISA9—shisa family member 9; 6. CRB3—crumbs cell polaritycomplex component 3; 7. AC118754.1—Arachidonate 15-lipoxygenase, ALOX15,Smoothelin- like protein 2, SMTNL2, Glutathione hydrolase 6, GGT6, Myb-binding protein 1A, MYBBP1A, Protein spinster homolog 2, SPNS2 8.ITGB6—integrin subunit beta 6; 9. CDH1—cadherin 1; 10. LSR—lipolysisstimulated lipoprotein receptor; 11. EPCAM—epithelial cell adhesionmolecule; 12. AJAP1—adherens junctions associated protein 1; 13.ANO9—anoctamin 9; 14. CLDN7—claudin 7; 15. EFNA1—ephrin A1; 16.MAL2—mal, T cell differentiation protein 2 (gene/pseudogene); 17.F11R—F11 receptor; 18. L1CAM—L1 cell adhesion molecule; 19. GFRA1—GDNFfamily receptor alpha 1; 20. IGSF3—immunoglobulin superfamily member 3;21. TNF—tumor necrosis factor; 22. MMP7—matrix metallopeptidase 7; 23.FOLR1—folate receptor alpha; 24. TGFA—transfonning growth factor alpha;25. C3—complement C3; 26. TNFSF10—TNF superfamily member 10; 27.PDGFB—platelet derived growth factor subunit B; and/or 28. WWC1—WW andC2 domain containing 1.

As will be understood by one of skill in the art, suitable combinationsof the markers listed in Table 4 may be used to separate kidney TAFcells from TAF-MSCs by selecting for specific markers from Table 4 orcombinations of two, three, four, five, six or more markers from Table4. In certain examples, kidney TAF MSCs can be more specificallyidentified by identifying a combination of stronger expression, such as12-fold or more stronger expression (optionally with TPMthreshold >2000) of any combination of the foregoing markers, e.g.,HAVCR1 and/or CD24 and/or CLDN6 and/or ABCB1 and/or SHISA9 and/or CRB3as compared to TAF-MSCs. When using combinations of markers,identification may be achieved with a lower threshold of strongerexpression, such as 4-fold or more, 6-fold or more, or 8-fold or moreexpression of each of the markers.

In contrast to the above surface markers that may be more stronglyexpressed on the surface of kidney TAF MSCs (positive markers), incertain examples, the below surface markers may be more weakly expressedon kidney TAF cells as compared to other cell types (negative markers),such as such as ⅛-fold or less expression (optionally with TPMthreshold >500) of any combination of the foregoing markers otherTAF-derived cells and/or TAF-MSC cells: GREM1, PDGFRB, BGN, FAP, CXCL12,CCKAR, CD248. When using combinations of negative markers,identification may be achieved with a lower threshold of weakerexpression, such as ½-fold or less, ¼-fold or less, or ⅙-fold or lessexpression of each of the markers.

Combinations of two or more these negative markers can also be used tomore specifically isolate kidney TAF MSCs. In addition, those skilled inthe art will also recognize that combinations including both negativeand positive markers, such as at any of the thresholds described above,can also be effective to more specifically isolate kidney TAF MSCs.

Skin TAF Cell Markers

Similar to the lung and kidney TAF MSC markers identified above, anumber of surface markers of interest associated with skin TAF cellswere identified. For example, a non-exclusive list of surface markersused to identify and separate skin TAF cells are provided below in Table5. The skin TAF MSC markers identified in Table 5 may have at least a12-fold increase in expression on prioritized clones compared to theaverage TAF-MSC clone (optionally with TPM threshold >2000). Moreover,as the number of different MSC-subtypes in TAF is limited, the selectionof the tissue specific MSC may be done by firstly characterization,thereafter a stepwise negative selection/sorting of the material bytaking into account the combined (multivariate) surface marker profileof the different tissue specific MSC's. One of skill in the art willunderstand that any such combination of these surface markers may beused for identifying and isolation of skin TAF cells from the generalpopulation of TAF-derived cells and/or TAF-MSC cells. In some examples,the below non-exclusive list of surface markers may be more highlyexpressed on the surface of skin-TAF cells as compared to other celltypes, such as other TAF-derived cells and/or TAF-MSC cells:

TABLE 5 1. TNFSF18—TNF superfamily member 18; 2. PCDH19—protocadherin19; 3. NCAM2—neural cell adhesion molecule 2; 4. TNFSF4—TNF superfamilymember 4; 5. CD248—Endosialin; 6. DDR2—discoidin domain receptortyrosine kinase 2; 7. HTR2B—5-hydroxytryptamine receptor 2B; 8.PCDH18—protocadherin 18; 9. SULF1—sulfatase 1; 10. MME—membranemetalloendopeptidase; 11. ADGRA2—adhesion G protein-coupled receptor A2;12. DCSTAMP—dendrocyte expressed seven transmembrane protein; 13.PDGFRA—platelet derived growth factor receptor alpha; 14. UNC5B—unc-5netrin receptor B; 15. SCUBE3—signal peptide, CUB domain and EGF likedomain containing 3; 16. CEMIP—cell migration inducing hyaluronidase 1;17. BDKRB1—bradykinin receptor B1; 18. FLT1—fms related tyrosine kinase1; 19. BDKRB2—bradykinin receptor B2; 20. FAP—fibroblast activationprotein alpha; 21. CASP1—caspase 1; and/or 22. SRPX2—sushi repeatcontaining protein X-linked 2.

As will be understood by one of skill in the art, suitable combinationsof the markers listed in Table 5 may be used to separate skin TAF MSCsfrom TAF-MSCs by selecting for specific markers from Table 5 orcombinations of two, three, four, five, six or more markers from Table5. In certain examples, skin TAF MSCs can be more specificallyidentified by identifying a combination of stronger expression, such as12-fold or more stronger expression (optionally with TPM>2000) of anycombination of the foregoing markers, e.g., TNFSF18 and/or PCDH19 and/orNCAM2 and/or TNFSF4 and/or CD248 and/or DDR2 as compared to TAF-MSCs.When using combinations of markers, identification may be achieved witha lower threshold of stronger expression, such as 4-fold or more, 6-foldor more, or 8-fold or more expression of each of the markers.

In contrast to the above surface markers that may be more stronglyexpressed on the surface of skin TAF cells (positive markers), incertain examples, the below surface markers may be more weakly expressedon skin TAF cells as compared to other cell types (negative markers),such as such as ⅛-fold or less expression (optionally with TPMthreshold >500) of any combination of the foregoing markers otherTAF-derived cells and/or TAF-MSC cells: CD24, TNFSF10, ITGB4, ABCB1.When using combinations of negative markers, identification may beachieved with a lower threshold of weaker expression, such as ½-fold orless, ¼-fold or less, or ⅙-fold or less expression of each of themarkers.

Combinations of two or more these negative markers can also be used tomore specifically isolate skin TAF MSCs. In addition, those skilled inthe art will also recognize that combinations including both negativeand positive markers, such as at any of the thresholds described above,can also be effective to more specifically isolate skin TAF MSCs.

Neural TAF Cell Markers

Similar to the lung, kidney, and skin TAF MSC markers identified above,a number of surface markers of interest associated with neural TAF cellswere identified. For example, a non-exclusive list of surface markersused to identify and separate neural TAF cells are provided below. Theneural TAF MSC surface markers identified in Table 6 may have at least a3-fold increase in expression on prioritized clones compared to theaverage TAF-MSC clone (optionally with TPM threshold >500). Moreover, asthe number of different MSC-subtypes in TAF is limited, the selection ofthe tissue specific MSC may be done by firstly characterization,thereafter a stepwise negative selection/sorting of the material bytaking into account the combined (multivariate) surface marker profileof the different tissue specific MSC's. One of skill in the art willunderstand that any such combination of these surface markers may beused for identifying and isolation of neural TAF cells from the generalpopulation of TAF-derived cells and/or TAF-MSC cells. In some examples,the below non-exclusive list of surface markers may be more highlyexpressed on the surface of neural-TAF cells as compared to other celltypes, such as other TAF-derived cells and/or TAF-MSC cells:

TABLE 6 1. HAVCR1—hepatitis A virus cellular receptor 1; 2.ACKR3—atypical chemokine receptor 3; 3. OSCAR—osteoclast associatedIg-like receptor; 4. C3—complement C3; 5. SIRPB1—signal regulatoryprotein beta 1; 6. SLC6A6—solute carrier family 6 member 6; 7.CCKAR—cholecystokinin A receptor; 8. TNFSF10—TNF superfamily member 10;9. CLSTN2—calsyntenin 2; 10. TENM2—teneurin transmembrane protein 2; 11.SFRP1—secreted frizzled related protein 1; 12.PIK3IP1—phosphoinositide-3-kinase interacting protein 1; 13.SCNN1D—sodium channel epithelial 1 delta subunit; 14. CLDN11—claudin 11;15. ALDH3B1—aldehyde dehydrogenase 3 family member B1; and/or 16.ITGB4—integrin subunit beta 4.

As will be understood by one of skill in the art, suitable combinationsof the markers listed in Table 6 may be used to separate neural TAF MSCsfrom TAF-MSCs by selecting for specific markers from Table 6 orcombinations of two, three, four, five, six or more markers from Table6. In certain examples, neural TAF MSCs can be more specificallyidentified by identifying a combination of stronger expression, such as3-fold or more stronger expression (optionally with TPM threshold >500)of any combination of the foregoing markers, e.g., HAVCR1 and/or ACKR3and/or OSCAR and/or C3 and/or SIRPB1 and/or SLC6A6 as compared toTAF-MSCs. When using combinations of markers, identification may beachieved with a lower threshold of stronger expression, such as 2-foldor more or a higher threshold such as 6-fold or more, 8-fold or more, or12-fold or more expression of each of the markers. In addition, thoseskilled in the art will also recognize that combinations including bothnegative and positive markers, such as at any of the thresholdsdescribed above, can also be effective to more specifically isolateneural TAF MSCs.

All of the features disclosed in this specification (including anyaccompanying exhibits, claims, abstract and drawings), and/or all of thesteps of any method or process so disclosed, may be combined in anycombination, except combinations where at least some of such featuresand/or steps are mutually exclusive. The disclosure is not restricted tothe details of any foregoing examples. The disclosure extends to anynovel one, or any novel combination, of the features disclosed in thisspecification (including any accompanying claims, abstract anddrawings), or to any novel one, or any novel combination, of the stepsof any method or process so disclosed.

Those skilled in the art will appreciate that in some examples, theactual steps taken in the processes illustrated or disclosed may differfrom those shown in the figures. Depending on the example, certain ofthe steps described above may be removed, others may be added. Forexample, the actual steps or order of steps taken in the disclosedprocesses may differ from those shown in the figure. Depending on theexample, certain of the steps described above may be removed, others maybe added. Furthermore, the features and attributes of the specificexamples disclosed above may be combined in different ways to formadditional examples, all of which fall within the scope of the presentdisclosure.

Conditional language, such as “can,” “could,” “might,” or “may,” unlessspecifically stated otherwise, or otherwise understood within thecontext as used, is generally intended to convey that certain examplesinclude, while other examples do not include, certain features,elements, or steps. Thus, such conditional language is not generallyintended to imply that features, elements, or steps are in any wayrequired for one or more examples or that one or more examplesnecessarily include logic for deciding, with or without user input orprompting, whether these features, elements, or steps are included orare to be performed in any particular example. The terms “comprising,”“including,” “having,” and the like are synonymous and are usedinclusively, in an open-ended fashion, and do not exclude additionalelements, features, acts, operations, and so forth. Also, the term “or”is used in its inclusive sense (and not in its exclusive sense) so thatwhen used, for example, to connect a list of elements, the term “or”means one, some, or all of the elements in the list. Likewise, the term“and/or” in reference to a list of two or more items, covers all of thefollowing interpretations of the word: any one of the items in the list,all of the items in the list, and any combination of the items in thelist. Further, the term “each,” as used herein, in addition to havingits ordinary meaning, can mean any subset of a set of elements to whichthe term “each” is applied. Additionally, the words “herein,” “above,”“below,” and words of similar import, when used in this application,refer to this application as a whole and not to any particular portionsof this application.

Conjunctive language such as the phrase “at least one of X, Y, and Z,”unless specifically stated otherwise, is otherwise understood with thecontext as used in general to convey that an item, term, etc. may beeither X, Y, or Z. Thus, such conjunctive language is not generallyintended to imply that certain examples require the presence of at leastone of X, at least one of Y, and at least one of Z.

Language of degree used herein, such as the terms “approximately,”“about,” “generally,” and “substantially” as used herein represent avalue, amount, or characteristic close to the stated value, amount, orcharacteristic that still performs a desired function or achieves adesired result. For example, the terms “approximately”, “about”,“generally,” and “substantially” may refer to an amount that is withinless than 10% of, within less than 5% of, within less than 1% of, withinless than 0.1% of, and within less than 0.01% of the stated amount. Asanother example, in certain examples, the terms “generally parallel” and“substantially parallel” refer to a value, amount, or characteristicthat departs from exactly parallel by less than or equal to 15 degrees,10 degrees, 5 degrees, 3 degrees, 1 degree, or 0.1 degree.

Various modifications to the implementations described in thisdisclosure may be readily apparent to those skilled in the art, and thegeneric principles defined herein may be applied to otherimplementations without departing from the spirit or scope of thisdisclosure. Thus, the disclosure is not intended to be limited to theimplementations shown herein, but is to be accorded the widest scopeconsistent with the principles and features disclosed herein. Certainexamples of the disclosure are encompassed in the claim set listed belowor presented in the future.

1. A method for obtaining term amniotic fluid mesenchymal stem cells(TAF MSCs) from term amniotic fluid, comprising: providing term amnioticfluid (TAF); removing particulate material from the TAF to obtainpurified TAF cells; performing adherence selection on the purified TAFcells to obtain TAF adherence cells; passaging the TAF adherence cellsto obtain a population of cells comprising the TAF MSCs; and selectingthe TAF MSCs from the population as cells that express at least oneGroup A surface marker selected from the group consisting of TBC1 domainfamily member 3K, allograft inflammatory factor 1 like, cadherin relatedfamily member 1, sodium/potassium transporting ATPase interacting 4, ATPbinding cassette subfamily B member 1, plasmalemma vesicle associatedprotein, mesothelin, L1 cell adhesion molecule, hepatitis A viruscellular receptor 1, mal, T cell differentiation protein 2(gene/pseudogene), SLAM family member 7, double C2 domain beta,endothelial cell adhesion molecule, gamma-aminobutyric acid type Areceptor beta1 subunit, cadherin 16, immunoglobulin superfamily member3, desmocollin 3, regulator of hemoglobinization and erythroid cellexpansion, potassium voltage-gated channel interacting protein 1, CD70molecule, GDNF family receptor alpha 1, crumbs cell polarity complexcomponent 3, claudin 1, novel transcript sodium voltage-gated channelalpha subunit 5, fibroblast growth factor receptor 4, potassium two poredomain channel subfamily K member 3, dysferlin, ephrin A1, potassiuminwardly rectifying channel subfamily J member 16, membrane associatedring-CH-type finger 1, synaptotagmin like 1, calsyntenin 2, integrinsubunit beta 4, vesicle associated membrane protein 8, G protein-coupledreceptor class C group 5 member C, CD24 molecule, cadherin EGF LAGseven-pass G-type receptor 2, cadherin 8, glutamate receptor interactingprotein 1, dematin actin binding protein, F11 receptor, cell adhesionmolecule 1, cadherin 6, coagulation factor II thrombin receptor like 2,LY6/PLAUR domain containing 1, solute carrier family 6 member 6,desmoglein 2, adhesion G protein-coupled receptor G1, cholecystokinin Areceptor, oxytocin receptor, integrin subunit alpha 3, adhesion moleculewith Ig like domain 2, cadherin EGF LAG seven-pass G-type receptor 1,and EPH receptor B2, thereby obtaining the TAF MSCs.
 2. The method ofclaim 1, wherein selecting TAF MSCs comprises selecting TAF MSCs thathave a reduced expression of markers selected from the group consistingof IL13RA2, CLU, TMEM119, CEMIP, LSP1, GPNMB, FAP, CRLF1, MME, CLMP,BGN, DDR2.
 3. The method of claim 1, wherein removing particulate mattercomprises filtering and centrifuging the TAF.
 4. The method of claim 1,wherein performing adherence selection on the purified TAF cellscomprises adhering the purified TAF cells to a surface coated with avitronectin-based substrate.
 5. The method of claim 1, wherein theselecting step is performed using fluorescence activated cell sorting(FACS).
 6. The method of claim 1, wherein the selecting step isperformed with antibodies directed to any of the markers or surfacemarkers.
 7. The method of claim 1, wherein the selecting step comprisesselecting TAF MSCs that express at least two markers from the Group Asurface markers.
 8. The method of claim 1, wherein the selecting stepcomprises selecting TAF MSCs that express at least three markers fromthe Group A surface markers.
 9. The method of claim 1, wherein theselecting step comprises selecting TAF MSCs that express at least fourmarkers from the Group A surface markers.
 10. The method of claim 1,wherein the selecting step comprises a plurality of sorting steps, eachsorting step comprising directing TAF MSCs into a first output group ora second output group in dependence on a set of markers expressed or notexpressed by the respective TAF MSCs.
 11. The method of claim 10,wherein the selecting step comprises: a first sorting step to direct TAFMSCs that express a Group A surface marker into a first output group,and a second sorting step to direct TAF MSCs from the first output groupthat express a second set of markers into a second output group.
 12. Amethod for obtaining term amniotic fluid lung mesenchymal stem cells(lung TAF MSCs) from term amniotic fluid, comprising: providing termamniotic fluid (TAF); removing particulate material from the TAF toobtain purified TAF cells; performing adherence selection on thepurified TAF cells to obtain TAF adherence cells; passaging the TAFadherence cells to obtain a population of cells comprising the lung TAFMSCs; and selecting the TAF lung MSCs from the population as cells thatexpress at least one Group B surface marker selected from the groupconsisting of PCDH19, DDR1, MME, IFITM10, BGN, NOTCH3, SULF1, TNFSF18,BDKRB1, FLT1, PDGFRA, TNFSF4, UNC5B, FAP, CASP1, CD248, DDR2, PCDH18,LRRC38, and CRLF1, thereby obtaining TAF lung mesenchymal stem cells.13. The method of claim 12, wherein selecting lung TAF MSCs comprisesexcluding MSCs that express a marker selected from the group consistingof CD24, ITGB4, TNFSF10, GFRA1, CD74, FGFR4, HAVCR1, and OSCAR.
 14. Themethod of claim 12, wherein the selecting step comprises selecting TAFMSCs that express at least two surface markers from the Group B surfacemarkers.
 15. The method of claim 12, wherein the selecting stepcomprises selecting TAF MSCs that express at least three surface markersfrom the Group B surface markers.
 16. The method of claim 12, whereinthe selecting step comprises selecting TAF MSCs that express at leastfour surface markers from the Group B surface markers.
 17. The method ofclaim 12, wherein the selecting step comprises selecting TAF MSCs thatexpress a surface marker selected from the group of CD248, DDR1, andLRRC38.
 18. The method of claim 17, wherein the selecting step comprisesselecting TAF MSCs that express CD248.
 19. The method of claim 18,wherein the selecting step comprises selecting TAF MSCs that expressCD248 in combination with a marker selected from the group of DDR1 andLRRC38.
 20. The method of claim 19, wherein the selecting step comprisesselecting TAF MSCs that express CD248, DDR1, and LRRC38.
 21. An isolatedpopulation of term amniotic fluid (TAF) mesenchymal stem cellsobtainable by the method according to claim 1, said cells expressing atleast one Group A surface marker selected from the group comprising ofTBC1 domain family member 3K, allograft inflammatory factor 1 like,cadherin related family member 1, sodium/potassium transporting ATPaseinteracting 4, ATP binding cassette subfamily B member 1, plasmalemmavesicle associated protein, mesothelin, L1 cell adhesion molecule,hepatitis A virus cellular receptor 1, mal, T cell differentiationprotein 2 (gene/pseudogene), SLAM family member 7, double C2 domainbeta, endothelial cell adhesion molecule, gamma-aminobutyric acid type Areceptor beta1 subunit, cadherin 16, immunoglobulin superfamily member3, desmocollin 3, regulator of hemoglobinization and erythroid cellexpansion, potassium voltage-gated channel interacting protein 1, CD70molecule, GDNF family receptor alpha 1, crumbs cell polarity complexcomponent 3, claudin 1, novel transcript sodium voltage-gated channelalpha subunit 5, fibroblast growth factor receptor 4, potassium two poredomain channel subfamily K member 3, dysferlin, ephrin A1, potassiuminwardly rectifying channel subfamily J member 16, membrane associatedring-CH-type finger 1, synaptotagmin like 1, calsyntenin 2, integrinsubunit beta 4, vesicle associated membrane protein 8, G protein-coupledreceptor class C group 5 member C, CD24 molecule, cadherin EGF LAGseven-pass G-type receptor 2, cadherin 8, glutamate receptor interactingprotein 1, dematin actin binding protein, F11 receptor, cell adhesionmolecule 1, cadherin 6, coagulation factor II thrombin receptor like 2,LY6/PLAUR domain containing 1, solute carrier family 6 member 6,desmoglein 2, adhesion G protein-coupled receptor G1, cholecystokinin Areceptor, oxytocin receptor, integrin subunit alpha 3, adhesion moleculewith Ig like domain 2, cadherin EGF LAG seven-pass G-type receptor 1,and EPH receptor B2.
 22. (canceled)
 23. (canceled)
 24. Isolated termamniotic fluid (TAF) mesenchymal lung stem cells obtainable by themethod according to claim 12, said cells expressing at least one Group Bsurface marker selected from the group consisting of PCDH19, DDR1, MME,IFITM10, BGN, NOTCH3, SULF1, TNFSF18, BDKRB1, FLT1, PDGFRA, TNFSF4,UNC5B, FAP, CASP1, CD248, DDR2, PCDH18 and CRLF1.
 25. (canceled) 26.(canceled)
 27. A method for obtaining term amniotic fluid kidneymesenchymal stem (kidney TAF MSCs) cells from term amniotic fluid,comprising: providing term amniotic fluid (TAF); removing particulatematerial from the TAF to obtain purified TAF cells; performing adherenceselection on the purified TAF cells to obtain TAF adherence cells;passaging the TAF adherence cells to obtain a population of cellscomprising the TAF kidney MSCs; and selecting the TAF kidney MSCs fromthe population as cells that express at least one Group C surface markerselected from the group consisting of HAVCR1, CD24, CLDN6, ABCB1,SHISA9, CRB3, AC118754.1, ITGB6, CDH1, LSR, EPCAM, AJAP1, ANO9, CLDN7,EFNA1, MAL2, F11R, L1CAM, GFRA1, IGSF3, TNF, MMP7, FOLR1, TGFA, C3,TNFSF10, PDGFB and WWC1, thereby obtaining TAF kidney MSCs. 28.(canceled)
 29. A composition comprising the isolated population of termamniotic fluid (TAF) kidney mesenchymal stem cells according to claim27.
 30. A method for obtaining term amniotic fluid skin mesenchymal stemcells (skin TAF MSCs) from term amniotic fluid, comprising: providingterm amniotic fluid (TAF); removing particulate material from the TAF toobtain purified TAF cells; performing adherence selection on thepurified TAF cells to obtain TAF adherence cells; passaging the TAFadherence cells to obtain a population of cells comprising the TAF skinMSCs; and selecting the skin TAF MSCs from the population as cells thatexpress at least one Group D surface marker selected from the groupconsisting of TNFSF18, PCDH19, NCAM2, TNFSF4, CD248, DDR2, HTR2B,PCDH18, SULF1, MME, ADGRA2, DCSTAMP, PDGFRA, UNC5B, SCUBE3, CEMIP,BDKRB1, FLT1, BDKRB2, FAP, CASP1, and SRPX2; and obtaining TAF skinMSCs.
 31. (canceled)
 32. (canceled)
 33. A method for obtaining termamniotic fluid neural mesenchymal stem cells (neural TAF MSCs) from termamniotic fluid, comprising: providing term amniotic fluid (TAF);removing particulate material from the TAF to obtain purified TAF cells;performing adherence selection on the purified TAF cells to obtain TAFadherence cells; passaging the TAF adherence cells to obtain apopulation of cells comprising the TAF neural MSCs; and selecting theTAF neural MSCs from the population as cells that express at least oneGroup E surface marker selected from the group consisting of HAVCR1,ACKR3, OSCAR, C3, SIRPB1, SLC6A6, CCKAR, TNFSF10, CLSTN2, TENM2, SFRP1,PIK3IP1, SCNN1D, CLDN11, ALDH3B1, and ITGB4; thereby obtaining TAFneural MSCs.
 34. (canceled)
 35. (canceled)
 36. (canceled)